The invention relates to novel retroviral gene transfer vectors, preferably expression vectors, which, because of a reduced content of viral genes, are distinguished by a higher safety standard and expression of non-viral nucleotide sequences in higher amounts.
The expression of foreign proteins in pro- and eukaryotic cells, i.e. of proteins which are usually expressed only in very small amounts, or not at all, in these cells, plays an essential role in research into the function of proteins, production of proteins and treatment of diseases. A prerequisite for expression of a foreign gene is that the genetic material which codes for the protein is introduced into the target cell. This is as a rule achieved with the aid of so-called vectors. Vectors are DNA molecules into which the DNA which codes for the protein to be expressed has been cloned and which contain DNA sequences which are of importance for expression of the protein.
For stable gene transfer into mammalian cells, retroviral vectors based on mouse leukaemia viruses (MLV) are currently the most frequently used and best characterized system (Miller, A. D. (1993) Methods Enzymol, 217; 581-599). Fields of use of retroviral vectors are, for example, over-expression of proteins with the aim of obtaining pure proteins, expression cloning with the aim of identifying new proteins, and stable expression of a protein in a body cell with the aim of a therapeutic action of the expressed protein. The level of transgene expression in the cell system relevant to the disease is decisive for the therapeutic potential of gene transfer here. For clinical use of gene transfer vectors, the safety of the vectors is furthermore of decisive importance. On the one hand, expression of viral gene products must be excluded here, since these may display a pathogenic action in the body. On the other hand, recombination with naturally occurring viruses should be ruled out, to prevent viral proteins being expressed under the control of the regulatory element introduced into the vector, or novel viruses with unknown properties being formed.
Retroviral vectors can exist in two forms, as proviral DNA or as vector RNA. Proviral double-stranded DNA is integrated in a stable manner in the genome of the target cell. From this, the so-called genomic vector transcript is read, which is built up like a cell mRNA (messenger RNA), and is packed in viral particles and transmits the genetic information of the retrovirus. After retroviral infection of a target cell, the genomic vector transcript is transcribed by reverse transcription into a new provirus and integrated in a stable manner into the genome of the cell (Miller, A. D. (1993) Methods Enzymol. 217: 581-599).
The provirus is flanked at the 5xe2x80x2- and at the 3xe2x80x2-end by xe2x80x9clong terminal repeatsxe2x80x9d (LTR), which include the regions U3, R and U5 (FIG. 1). The U3 region contains enhancer/promoter sequences which control transcription of the vector. The R region carries the polyadenylation signal. The U5 region contains sequences which are necessary for integration of the retrovirus. The coding sequences of the exogenous protein are usually between 5xe2x80x2- and 3xe2x80x2-LTR in the vector and are flanked by control sequences of regulatory importance which are essential for the progress of the retroviral life cycle and at the same time influence the half-life of the RNA and the translation efficiency of the exogenous protein. The transcription start of the RNA lies at the boundary of the R region of the 5xe2x80x2-LTR. The transcription end is determined by polyadenylation and termination signals in the R region of the 3xe2x80x2-LTR. A polyadenosine tail is attached at the end of the R region of the 3xe2x80x2-LTR. Splicing signals of the retrovirus/vector can lead to transcripts with internal deletions.
The 5xe2x80x2-untranslated region of the retroviral vector is composed of (A. D. Miller (1993) loc. cit.):
R region and U5 region of the 5xe2x80x2-LTR (approx. 150 nucleotides, necessary for reverse transcription and integration)
Primer binding site (18 nucleotides, necessary for reverse transcription)
Leader region (in current retroviral vectors at least 800 nucleotides long). This follows the primer binding site and extends to the start of the coding sequence. The leader region contains the packing and dimerization signal, which is necessary for incorporation of the RNA into retroviral particles. At the start of the leader lies the retroviral splicing donor, and at the end of the leader there can be a cryptic (i.e. recognized only in a portion of the transcripts) splicing acceptor. The splicing donor and splicing acceptor determine the start and end of the RNA sequences which can be removed from the primary transcript by the splicing operation even before export into the cytoplasm. The efficiency of the splicing reaction depends on the suitability of the splicing signals. In addition to the sequences of the splicing donor and splicing acceptor, the polypyrimidine tract lying before the splicing acceptor and the subsequent so-called branch site (see below) determine the efficiency of the splicing (Zhuang, Y. A. et al. (1989) Proc. Natl. Acad. Sci. USA, 86: 2752-2756).
The efficiency of the translation is impaired by the long length of the 5xe2x80x2-untranslated region (5xe2x80x2-UTR). The splicing signals of the leader can give rise to shortened transcripts with significantly shorter 5xe2x80x2-UTR, which have an increased translation efficiency (Armentano, D. et al. (1987) J. Virol. 61: 1647-1620; Bender, M. A. et al. (1987) J. Virol. 61: 1639-1646; Krall, W. J. et al. (1996) Gene Ther. 3: 37-48).
The 3xe2x80x2-untranslated region of the RNA contains the polypurine tract, which is necessary for the reverse transcription, and the U3 and R region of the 3xe2x80x2-LTR.
After the retroviral life cycle, the U3 region of the 3xe2x80x2-UTR is copied into both LTRs. If the U3 region contains enhancer/promoter regions, these control the transcription of the vector-RNA in the infected cell (Baum, C. et al. (1995) J. Virol. 69: 7541-7547). The end of the RNA is the polyadenosine tail of approx. 200 nucleotides, which co-determines the stability in the cytoplasm.
In addition to the control elements, retroviruses contain nucleotide sequences which code for viral proteins. These include the gag gene, which codes for structure proteins of the virus, the pro gene, which codes for the virion protease, the pol gene, which codes for the reverse transcriptase, and the env gene, which codes for virus envelope glycoproteins. If the retroviral vector does not contain one of these genes, it is replication-incompetent. To produce infectious virus particles from a replication-incompetent retroviral vector, a helper virus and/or a packing-competent cell line which provide the properties lacking from the retroviral vector are required.
The viral sequences contained in the retroviral vectors can give rise to recombination with complementary retroviruses in the packing cell, as a result of which replication-competent retroviruses, which can induce leukaemias and encephalopathies in animal studies, can form (Anderson, W. F. (1993) Hum. Gene Ther. 4; 311-321; Mxc3xcnk, C. (1997) PNAS 94: 5837-5842). The residual gag and pol genes furthermore contain a large number of cryptic reading frames, i.e. nucleotide sequences which code for an amino acid sequence in a reading frame other than for the actual gene, but in the normal case are not read, since the control elements and/or the start codon are missing. Nevertheless, it cannot be ruled out that immunogenic or toxic peptides can be generated in the target cell by the open reading frame.
The target cell of the retroviral expression vector is transduced with infectious virus particles. These particles are produced with the aid of a packing-competent cell line. It is important here that as many infectious virus particles as possible per ml of cell culture supernatant are produced by the packing-competent cells, that is to say that the virus titre, stated in infectious units per ml of culture medium, is as high as possible.
Retroviral vectors are often produced on the basis of control elements which are taken from mouse laukaemia viruses (MLV) (Miller, A. D., loc. cit.). These vectors are called MLV-based vectors. A central field of use of this group of retroviral vectors is gene transfer in haematopoietic stem cells (Dunbar, C. E. (1996) Ann. Rev. Med. 47: 11-20). Therapeutic actions are said to be achieved here with the by transfer and expression of the protein coded by the vector. The protein is produced permanently in the cells. Long-lasting treatment which acts directly on the genetic defects on which the disease is based is therefore conceivable. For example, the vector can carry genes such as human mdr1 (multidrug resistance 1) to protect the stem cells against the toxic side effects of a cytostatic drug treatment (stem cell protection) (Baum, C. et al, (1996) Gene Ther. 3: 1-3, Baum, C. et al, (1995) loc. cit.).
The prior art describes retroviral vectors in which the gag gene including a 60 bp sequence of the 5xe2x80x2-UTR lying directly 5xe2x80x2 (i.e. upstream) of the start codon of the gag gene has been removed. However, only very low virus titres have been obtained with these vectors. Works by Armentano and Bender from 1987 (Armentano, D. et al. (1987) J. Virol. 61: 1647-1650); Bender, M. A. et al. (1987) J. Virol. 61: 1639-1646) have shown that the virus titre of retroviral vectors derived from MLV is higher if some of the gag gone (at least 400 bp) is left in the vector. It is concluded from this that the packing signal decisive for the retroviral titre is not limited only to the 5xe2x80x2-untranslated region of the retrovirus, but extends into the viral gag sequence. This opinion has since also been expressed in textbooks (Kriegler, M. (1990) Gene Transfer and Expression, Stockton Press, New York, p. 52). Por this reason, no retroviral vectors which contain fewer than 400 bp of the gag gene or in which the gag sequences have been removed completely are produced in the prior art. The vectors corresponding to the prior art are also called (gag+) vectors.
In retroviruses, the translation level of retroviral sequences depends on the presence of appropriate splicing signals and the position of the start codon in the spliced RNA. A splicing signal is understood as a nucleotide sequence which regulates splicing of the RNA formed during the transcription. These splicing signals consist of different nucleotide sequences, depending on the virus strain on which the vector is based. However, a splicing signal originating from a certain virus is as a rule also active in vectors which are derived from other viruses (Bowtell, D. D. et al. (1988) J. Virol. 62: 2464-2473). All splicing signals have a common consensus sequence which contains a xe2x80x9cbranch sitexe2x80x9d. The nucleophile which is necessary for the first transesterification step of the splicing reaction is designated the xe2x80x9cbranch sitexe2x80x9d. An denosine nucleotide usually functions as the xe2x80x9cbranch sitexe2x80x9d. In viruses derived from MLV, the translation level of retroviral env sequences depends on the presence of appropriate splicing signals and the position of the start codon in the spliced RNA, This observation was the basis of the development of MFG vectors (Riviere, I. et al. (1995) PNAS 92: 6733-6737). The abbreviation MFG is derived from a proper name which is not explained in the literature (Dranoff, G. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3539-3543). MFG vectors are built up as (gag+) vectors and additionally also contain a fragment of the retroviral pol gene which carries the Moloney MLV splicing acceptor. The coding sequence of the gene to be transferred is placed exactly at the site and as a substitute of the retroviral env gene (FIG. 1). Compared with other MLV vectors with identical enhancer-promoter sequences, MFG vectors show an increased expression of the exogenous gene (Krall, W. J. et al. (1996) Gene Ther. 3: 37-48). However, not more than 50% of the transcripts produced are spliced in MFG vectors, so that a high proportion of the transcripts is still lost for the translation. MFG vectors also show problems in respect of the safety aspect, since they carry high proportions of the viral gag and pol genes to achieve a high packing efficiency and good splicing rates. These viral sequences can give rise to recombination with complementary retroviruses in the packing cell (see above). MFG vectors are therefore of only limited suitability for experimental use and not very suitable for therapeutic use, since they carry safety risks which are too high because of the immunogenic or toxic potential.
Another group of MLV-based retroviral vectors are the LX vectors. LX vectors Are also (gag+) vectors. LX means LTR gene x (Miller, A. D. and G. J. Rosman (1987) Biotechniques 7: 980-990). In LX vectors, the transgene is incorporated centrally into the gag region (approx. 400 base pairs 3xe2x80x2 of the gag start codon, which has been mutated to a stop codon). The other gag, pol and env sequences have been removed completely. The LX vectors thus carry fewer viral sequences than the MFG vectors. Nevertheless, the efficiency of the splicingxe2x80x94and therefore also of the translation of the transgenexe2x80x94with the LX vectors is as a rule lower than with the MFG vectors (Armentano, D. et al. (1987) J. Virol. 61: 1647-1620; Bender, M. A. et al. (1987) J. Virol. 61: 1639-1646; Krall, W. J. et al. (1996) Gene Ther. 3: 37-48).
LX or MFG vectors which, in addition to the nucleotide sequences which code for the exogenous proteins, also contain sequences for residual viral gene products and at least 400 bp of the gag gene have hitherto found use in the prior art. As already mentioned, this results in the disadvantage that with these vectors there is always the risk that viral proteins or peptides are also expressed and recombination with other viruses takes place. Furthermore, the minimum of 400 bp of the gag gene reduce the cloning capacity, i.e. the maximum length of the exogenous nucleotide sequence of the vector, since a retroviral vector can comprise not more than 10 kb. Another disadvantage of MFG or LX vectors lies in the fact that at least half the transcripts are not spliced and are therefore not available for translation.
An object of the present invention is therefore to provide retroviral vectors which do not have the disadvantages mentioned for the vectors used in the prior art. In particular, it is an object of the present invention to provide vectors which meet a higher safety standard than the vectors known to date. In particular, the risk of viral proteins being generated in the target cells should be minimized; preferably, no immunogenic or toxic proteins should be expressed. An essential aim is to reduce or rule out completely the probability of recombination with other viruses. It is furthermore an object of the invention to provide retroviral expression vectors which have a comparable virus titre and a higher cloning capacity with respect to the vectors used in the prior art and allow expression of non-viral nucleotide sequences in large amounts.
According to the invention, the object is achieved in that for the first time retroviral vectors which contain less than 400 bp of the nucleotide sequence which codes for gag are provided. The vectors according to the invention preferably contain less than 200 bp or less than 80 bp of a nucleotide sequence which codes for gag. According to a preferred embodiment of the present invention, the retroviral vectors contain no sequences or sequence sections which code for gag. Vectors in which the sequences which code for viral proteins are removed completely are particularly preferred. The retroviral vector is preferably used as an expression vector.
The invention thus relates to a retroviral vector which contains a nucleotide sequence N of the general formula
5xe2x80x2-end-[5xe2x80x2-UTR]-[gag]-[ex]-[vir]-3xe2x80x2-end 
wherein
[5xe2x80x2-UTR] represents a non-coding nucleotide sequence which lies in retroviruses directly 5xe2x80x2, i.e. upstream, of the start codon of the gag gene,
[gag] represents a section of the nucleotide sequence which codes for the gag gene,
[ex] represents a non-viral nucleotide sequence and
[vir] represents a retroviral nucleotide sequence,
and which is characterized in that
the number of nucleotides in [gag] is less than 400 bp.
In the context of the present invention, it has been found, surprisingly, that in contrast to prevailing opinion, high virus titres can he achieved with the construction according to the invention, which contains less than 400 bp of the gag gene and according to a particularly preferred embodiment no nucleotides which code for the gag gene, and a high expression of the non-viral nucleotide sequences is possible. By reducing the gag content, the cloning capacity and the safety of the vectors according to the invention are increased.
Possible [5xe2x80x2-UTR] are all nucleotide sequences which, in retroviruses or retroviral constructions derived from retroviruses, lie directly 5xe2x80x2, i.e. upstream, of the start codon of the gag gene, where, in the retroviral constructions, these nucleotide sequences should not be changed such that the change leads to a significant (more than one order of magnitude) reduction in the virus titre or translation efficiency compared with the non-changed sequence. Mutations and relatively small deletions which do not lead to a reduction in the virus titre or translation efficiency are included according to the invention. A deletion of a sequence section comprising at least 60 bp which lies directly before the start codon of the gag gene is excluded according to the invention. According to the invention, xe2x80x9cdirectly before the start codonxe2x80x9d means that apart from incorporated splicing acceptor nucleotides and/or polylinker fragments, there are no further nucleotides between this sequence section and the start codon of the gag gene.
According to a particular embodiment of the invention, the [5xe2x80x2-UTR] contains at least one sequence section which represents a splicing acceptor. The splicing acceptor can originate from the FMEV vector SF1MSN, but also from other retroviral or non-retroviral vectors, viruses such as, for example, HIV, or also from intron-containing eukaryotic genes. The splicing acceptor here can be a large genomic restriction fragment of the viral pol gene. A synthetically produced oligonucleotide which represents only the minimum consensus for the splicing acceptor (including the so-called branch site) and has a length of 10-100, preferably 10-50, particularly preferably 20-50 base pairs is particularly preferred.
In another preferred embodiment, [5xe2x80x2-UTR] contains at no site the nucleotide triplet AUG (adenine-uracil-guanine), which serves as the start codon in retroviruses. As a result, translation of cryptic open reading frames in [5xe2x80x2-UTR] is ruled out. This also leads to an increase in the safety standard. The AUG-deleted nucleotide sequences are obtainable, for example, by a mutation by means of xe2x80x9csite-directed mutagenesisxe2x80x9d (see example 4).
According to the invention, 5xe2x80x2-UTR can contain a leader sequence. According to a particular embodiment, this can originate from the MESV virus (Grez, M. et al., Proc. Natl. Acad. Sci. USA 87: 9202-9206 (1990)), but it is also conceivable to use the leader sequence of MoMoLV or MoMuSV (Miller, A. D. and Rosmann, G. J., loc. cit.). The viruses of which the 5xe2x80x2-leaders can be used in the retroviral vector according to the invention furthermore include, for example, xe2x80x9cspleen necrosis virusxe2x80x9d (Olson P. et al. (1994) J. Virol. 68: 7060-7066) or xe2x80x9cHarvey murine sarcoma virusxe2x80x9d (Berlioz, C. et al. (1995) J. Virol. 69: 6400-6407), but are not limited to these.
The sequence element [gag] can be taken from any desired sections of the nucleotide sequence which codes for the gag protein. According to the invention, [gag] contains less than 400 bp. Preferably, [gag] contains less than 200 bp, preferably less than 80 bp.
According to a particularly preferred embodiment of the invention, the retroviral expression vector 5xe2x80x2 of [ex] contains no nucleotides or sequence fragments which originate from the gag gene. This means that the non-viral nucleotide sequence [ex] lies directly 3xe2x80x2 of [5xe2x80x2-UTR]. According to the invention, xe2x80x9cdirectly 3xe2x80x2 of [5xe2x80x2-UTR]xe2x80x9d means that apart from incorporated splicing acceptor nucleotides and/or polylinker fragments, there are no further nucleotides between this sequence section and the first nucleotide of [ex]. The non-viral nucleotide sequence [ex] is then incorporated at the site of the gag gene into the retroviral expression vector. As a result, the architecture of a native retrovirus is reconstructed. This accurate imitation of the structure of the native retrovirus leads to an increased expression of the gene, which can be increased further by introducing a synthetic splicing acceptor.
This particularly preferred embodiment, in which [ex] replaces the gag gene, can be obtained by removing the residual gag contents by means of PCR (see example 1). These techniques are known to the expert (Ausubel, I. et al. (1994) Current Protocols in Molecular Biology. John Wiley and Sons, New York). This leads to a vector which carries a xe2x80x9cmultiple cloning sitexe2x80x9d instead of the gag gene. A multiple cloning site is a nucleotide sequence which contains various cutting sites for restriction enzymes. The desired non-viral nucleotide sequence [ex] can then be cloned directly on the 3xe2x80x2-end of the [5xe2x80x2 region] by means of techniques known to the expert.
According to the invention, the nucleotide sequence [ex] is between [gag] and [vir] and represents a non-viral nucleotide sequence. Preferably, it contains at least one nucleotide sequence which codes for a non-viral protein. The sequence [ex] can also comprise a non-viral RNA sequence. According to one embodiment of the invention, the non-viral nucleotide sequence [ex] contains a nucleotide sequence which codes for the MDR1 gene. According to another embodiment, [ex] contains a nucleotide sequence which codes for enhanced humanized green fluorescent protein, which is suitable as a cytoplasmic marker (EGFP; Cormack, B. P. et al. (1996) Gene 173: 33-38).
Other sequences which are suitable according to the invention include, but without being limited to the examples mentioned:
genes which, like EGFP, are used as cell markers (e.g. surface proteins such as human low affinity nerve growth factor receptor (Fehse, B. et al. (1997) Hum. Gene Ther. 8: 1815-1824))
genes which, like MDR1, can impart resistance to cytotoxic drugs (e.g. dehydrofolate reductase; Zhao, S. C. et al. (1997) Hum. Gene Ther. 8: 903-909).
genes which can correct congenital metabolic diseases (e.g. a-L-iduronidase for treatment of Hurler-Scheie syndrome; Huang, M. M. et al. (1997) Gene Ther. 4: 1150-1159).
Since the nature of the gene has no influence on the improvement according to the invention of the vectors, potentially all sequences which are transferred into retroviral vectors are of interest as [ex]. In the preferred embodiment of the invention, the cloning capacity for the genes to be transferred is increased by approx. 400 base pairs by elimination of all virus-coding sequences.
The nucleotide sequences which code for non-viral proteins can be cloned into the non-viral nucleotide sequence [ex] either in the reading direction, i.e. in the 5xe2x80x2xe2x86x923xe2x80x2 direction, or in the opposite orientation, i.e. in the 3xe2x80x2xe2x86x925xe2x80x2 direction. According to a particular embodiment, the first nucleotide of the start codon of the nucleotide sequence which codes for the non-viral protein represents the 5xe2x80x2-end of [ex]. In addition to the region which codes for proteins, the non-viral nucleotide sequence [ex] can furthermore comprise regulatory nucleotide sequences. If the nucleotide sequence [ex] contains several nucleotide sequences which code for non-viral proteins, these nucleotide sequences can either follow one another directly or be separated from one another by regulatory nucleotide sequences. According to the invention, regulatory nucleotide sequences here are to be understood as meaning all nucleotide sequences which can influence the expression of the non-viral proteins. These include, in particular, splicing acceptor sites. Further possibilities for linking transcription units in [ex] are internal ribosome entry signals, internal promoters or fusion genes (Hildinger, M. et al. (1998) Hum. Gene Ther. 9: 33-42).
Important regulatory nucleotide sequences are furthermore promoter and enhancer sequences. The promoter and enhancer sequences can originate from any desired viral or non-viral genes. The promoters preferably originate from MLV (Baum, C. et al. (1995) J. Virol. 69: 7541-7547), but are not limited to this. Promoter and enhancer sequences which are specific for the particular target cell are particularly suitable because a cell-specific expression is possible in this way.
The promoter and enhancer sequences can lie both at the 5xe2x80x2-end and at the 3xe2x80x2-end of [ex]. They can likewise lie at any position in [ex], including within the nucleotide sequences which code for non-viral proteins.
According to another embodiment of the invention, the nucleotide sequences which code for non-viral proteins contain those mutations which, without changing the protein sequence coded by the non-viral nucleotide sequence, lead to removal of cryptic splicing sites and/or cryptic poly-A sites. This avoids the mRNAs formed by transcription of the non-viral nucleotide sequences which code for the proteins being incorrectly spliced or too short if the poly-A tail is attached too early (McIvor, R. S. (1990) Virology 176: 652-655; Johnson, J. J. et al. (1995) Hum. Gene Ther. 6: 611-623). Site-directed mutagenesis is the currently customary technique preferred by the expert for introduction of mutations. According to the invention, however, other techniques can also be used. Other mutations which are of interest according to the invention comprise removal of ATG triplets, which can lead to a defective start to the translation of the sequence in [ex], improvement of the translation start of the sequence in [ex] by optimization of the so-called Kozak consensus sequence (Krall, W. J. et al. (1996) Gene Ther. 3: 37-48), or introduction of signals which lead to an improved nucleus export of the RNA (Pasquinelli, A. E. et al. (1997) EMBO J. 16: 7500-7510); the latter can also be incorporated into the vector at a site other than in [ex].
According to the invention, [vir] represents a retroviral sequence which can contain nucleotide sequences which code for one or more viral proteins or for parts thereof. In the context of the present invention, the sequence sections which code for viral proteins are preferably reduced in number and length in the retroviral vector. In particular, the vector preferably contains no sequences or sequence sections which code for the pol protein, the pro protein, the env protein and/or other viral proteins.
According to the invention, an expression vector which contains no nucleotide sequences which code for the gag protein, particularly preferably none which code for the gag protein or for other viral proteins, is preferred. This means that the retroviral vector overall contains no nucleotide sequences which code for residual viral proteins. A retroviral expression vector which both contains no sequence sections which code for residual viral proteins or for parts thereof and contains the nucleotide triplet AUG at no site apart from in [ex] is especially preferred. This ensures that both no residual viral proteins or parts thereof and no cryptic peptides are produced. The exclusion of nucleotide sequences which code for viral proteins ensures that the risk of recombination, based on sequence homology, with MLV sequences in the packing cell is minimized. The risk of an aberrant translation of viral peptides in the target cell is also reduced. Furthermore, the safety standard is increased in that the retroviral vector contains no start codon with the sequence AUG.
Downstream of the nucleotide sequence N, i.e. 3xe2x80x2, is in general a 3xe2x80x2-LTR in the vectors according to the invention. According to a particular embodiment, the 3xe2x80x2-LTR from spleen focus forming virus (SFFVp) can be used. The viruses of which the 3xe2x80x2-LTR can be used furthermore include other MLV, such as Friend-MLV, MPSV or MOMLV (Baum, C. et al. (1995) j. Virol. 69: 7541-7547), but are not limited to these.
According to a particularly preferred embodiment, the retroviral vectors according to the invention are the vectors SFxcex271m4 (DSM 12066) and SFxcex291 mSA1 (DSM 12065), deposited on Mar. 20, 1998, under conditions of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zelikulturen [German Collection of Microorganisms and Cell Cultures], Mascheroder Weg 1b, 38124 Braunschweig, or vectors derived therefrom.
The vector deposited under DSM 12065 is distinguished in that all the nucleotide sequences which code for viral proteins have been removed. At the 5xe2x80x2-end of the proviral DNA are the U3, R and U5 regions of the 5xe2x80x2-LTR. The CAP site at which the transcription of the viral mRNA starts lies in the 5xe2x80x2-LTR. 3xe2x80x2 of 5xe2x80x2-LTR lies a nucleotide sequence which contains a splicing acceptor (SA), a packing region (xcexa8) and a splicing donor (SD). Nucleotide 271 (counted from the CAP site) has been mutated to remove a cryptic AUG. At the site at which the sequences which code for the gag gene were to be found is the variant mSA1 of the MDR1 gene, which is distinguished in that a cryptic splicing acceptor has been mutated at position 2320 (counted from the start codon of the MDR1 gene). 3xe2x80x2 of the MDR1 variant mSA1 is the 3xe2x80x2-LTR with the U3, R and U5 regions (cf. FIG. 2).
The vector deposited under DSM 12066 is distinguished in that all the nucleotide sequences which code for viral proteins have been removed. At the 5xe2x80x2-end of the proviral DNA are the U3, R and US regions of the 5xe2x80x2-LTR. The CAP site at which transcription of the viral RNA starts lies in the 5xe2x80x2-LTR. 3xe2x80x2 of the 5xe2x80x2-LTR lies a nucleotide sequence which contains a splicing acceptor (SA), a packing region (xcexa8) and a splicing donor (SD). At the site at which the sequences which code for the gag gene were to be found is the variant m4 of the MDR1 gene, which is distinguished in that a cryptic splicing donor has been mutated at position 339 (counted from the start codon of the MDR1 gene), a cryptic splicing acceptor has been mutated at position 2320 and a cryptic poly(A) signal has been mutated at position 3303. 3xe2x80x2 of the MDR1 variant m4 is the 3xe2x80x2-LTR with the U3, R and U5 regions (cf. FIG. 2).
According to the invention, a vector which is derived from the vector deposited under DSM 12066 and in which a cryptic AUG has additionally been mutated at position 271 (counted from the CAP site) is furthermore preferred.
The last three vectors mentioned, i.e. the vectors deposited under DSM 12065 and 12066 and the vector derived from DSM 12066, are particularly preferably suitable for transfection of haematopoietic stem cells in the context of gene therapy in order to achieve resistance of the haematopoietic stem cells during chemotherapy (see below).
The present invention furthermore relates to a process for the preparation of an infectious virus particle. The techniques on which the process is based are known to the expert (Ausubel, I. et al. (1994) Current Protocols in Molecular Biology, John Wiley and Sons, New York). With the aid of a packing-competent cell line and/or a helper virus, infectious virus particles which contain the retroviral vector according to the invention are prepared. The helper virus is produced by the packing cells as a replication-competent particle.
The genome of the helper virus has been changed by genetic engineering so that the RNA of the helper virus which codes for the virus protein cannot be packed into the virus particles (Miller, A. D. et al. (1993) Methods Enzymol. 217: 581-599).
The present invention furthermore relates to a host cell transfected with the retroviral vector according to the invention. The host cell is preferably infected with an infectious virus particle (see above) which contains the retroviral vector according to the invention. This host cell can be chosen from the available immortalized haematopoietic cell lines (Baum, C. et al. (1995) J. Virol. 69: 7541-7547) or primary human blood cells (Eckert, H. G. et al. (1996) Blood 88: 3407-3415), but is not limited to these. Preferably, the host cell is a K-562 cell (human erythroleukaemia cell; Lozzio, C. B. et al. (1976) Cancer Res. 36: 4657-4662), which is transfected with the vector SFxcex271m4 (DSM deposit number DSM 12066) or with the vector SFxcex291mSA1 (DSM deposit number DSM 12065).
The invention furthermore relates to a process for the preparation of proteins, in which a host cell which has been transfected with a retroviral vector according to the invention or a virus particle according to the invention is cultured in a suitable medium under conditions which are necessary for expression of the retroviral proteins for which the nucleotide sequences in the nucleotide sequence [ex] code. These conditions can include culture in standard cell culture media, such as Delbecco""s modified essential medium (DMEM), which are supplemented with animal serum (for example foetal calf serum) (Ausubel et al., loc. cit.). Thereafter, the protein produced is purified with the aid of techniques known to the expert (Ausubel et al., loc. cit.), by separating it off from the cells and the medium.
According to the invention, the retroviral vector can be used therapeutically. This means that it is a constituent of a pharmaceutical preparation which additionally comprises pharmaceutically tolerated auxiliaries and/or excipients.
According to a particular embodiment of the invention, the retroviral gene transfer or expression vector is used in gene therapy. This includes the vector being present in a pharmaceutical preparation such that it can be introduced into the target cell. The retroviral vector according to the invention is preferably present packed in an infectious virus particle. The retroviral vector is preferably used for transfection of haematopoietic stem cells (Baum, C. et al. (1997) in: Concepts in Gene Therapy (M. Strauss, W. Barranger, eds.), De Gruyter, Berlin, p. 233-266).
The retroviral vector according to the invention is furthermore used for expression cloning of genes. For this, a cDNA library which contains the gene sought is cloned into the retroviral expression vector according to the invention. After packing of the vector in infectious particles and infection of host cells with the particles, the host cell which expresses the protein sought and therefore contains the gene sought can be detected, for example by means of antibodies or via functional tests.
According to the invention, the retroviral vector can furthermore be used for expression and/or over-expression of proteins or of RNA. For this purpose, the gene to be expressed or the RNA is cloned into the retroviral vector according to the invention. After packing in infectious virus particles and infection of a suitable host cell with the particles, the desired protein can be produced by known processes.
The invention is explained below with the aid of examples, figures and sequence protocols.